Methods and formulations for reducing reconstitution time of lyophilized polypeptides

ABSTRACT

The present invention relates to methods and formulations useful for reducing the reconstitution time of lyophilized polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional applicationSer. No. 16/420,974, filed May 23, 2019, which is continuation ofInternational Patent Application No. PCT/US2017/068024, filed Dec. 21,2017, which claims priority to and the benefit of U.S. ProvisionalApplication No. 62/438,232, filed Dec. 22, 2016, each of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and formulations useful forreducing the reconstitution time of lyophilized polypeptides.

BACKGROUND OF THE INVENTION

Biological molecules for pharmaceutical or other uses are oftenlyophilized, a process in which water is removed from a liquidcomposition after it is frozen and placed under vacuum. In general,lyophilization improves stability of a polypeptide composition by theremoval of water (as many biologic chemical degradations are hydrolytic)and by decreasing the overall mobility of the system (as dynamicmovement of sidechains and molecules is necessary for chemical andphysical degradation events to occur). Lyophilized polypeptides aresubsequently reconstituted prior to use, often in the very samecontainers or vials in which they were lyophilized and stored. Shortreconstitution time is preferable, especially in the context ofreconstitution of a lyophilized medicinal polypeptide composition (e.g.,an antibody composition).

High-concentration and high-dose (e.g., gram quantities) of lyophilizedpharmaceutical products pose unique processing and handling challenges,including long lyophilization cycles and often long reconstitutiontimes. (See, e.g., American Pharmaceutical Review, 13 (2010), pp. 31-3234-38.) One approach to controlling reconstitution time for biologicalmolecules is to decrease the protein concentration, thereby increasingthe fill volume. However, this approach can be unsuitable for high-doseformulations where increasing fill volume would necessitate the use ofmultiple vials per dose. In such cases, alternative lyophilizationformulations and methods are required.

The presence of tert-butyl alcohol (TB A) in lyophilization formulationshas been shown to improve reconstitution time of small quantities orlow-doses (e.g., milligram quantities) of biological molecules (See,e.g., U.S. Patent Application Publication No. US 2014/0044717; Nail etal., (2002) J Pharma Sci. Vol. 91; Degobert et al., (2016) DryingTechnology; Yong et al., (2009) Int J Pharmaceutics, 371:71-81;Teagarden and Baker (2002) Eur J Pharma Sci 15:115-133; Vessot andAndrieu (2012) Drying Technology (2012) 30:377-385).

However, the use of TBA in a co-solvent system in lyophilizedformulations of biological molecules and its effects on reconstitutiontime and polypeptide stability has not been well-characterized inpolypeptide formulations containing large quantities of protein (e.g.,gram quantities) or with high protein concentrations.

Thus a need exists for methods and formulations of lyophilizedbiological molecules for large quantities and concentrations (i.e.,high-concentration, high-dose) polypeptide compositions, includingantibody compositions, which provide reduced or fast reconstitutiontimes as well as maintain integrity and stability of the biologicalmolecule. The present invention meets this need by providing methods andformulations for reducing reconstitution times of lyophilizedpolypeptides, in which the methods and formulations maintain integrityand stability of the lyophilized polypeptides, in particular forlyophilized antibody compositions of high-concentration and/or high-doseformulations.

SUMMARY OF THE INVENTION

The present invention provides methods and formulations useful forreducing the reconstitution time of lyophilized polypeptides, forexample, lyophilized antibody compositions.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized polypeptide composition,wherein the method comprises preparing a liquid composition comprisingthe polypeptide, adding tert-butyl alcohol (TBA) to the liquidcomposition to form a liquid polypeptide/TBA mixture, freezing theliquid polypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBAmixture to form a lyophilized polypeptide composition, andreconstituting the lyophilized polypeptide composition with a diluent,wherein the time for reconstituting the polypeptide lyophilized in thepresence of TBA is less than the time for reconstituting the same amountof the same polypeptide lyophilized in the absence of TBA. In someembodiments, the amount of TBA added to the liquid polypeptidecomposition to form a liquid polypeptide/TBA mixture is between about0.5% to about 20% by volume. In other embodiments, the amount of TBAadded to the liquid polypeptide composition to form the liquidpolypeptide/TBA mixture is selected from the group consisting of 0.5%,1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, and 20% by volume. In yetother embodiments, the amount of TBA added to the liquid polypeptidecomposition to form the liquid polypeptide/TBA mixture is between about1% to about 20% by volume, about 1% to about 10% by volume, about 1% toabout 5% by volume, about 5% to about 20% by volume, or about 5% toabout 10% by volume. In one embodiment, the TBA is added to the liquidcomposition comprising the polypeptide immediately prior to freezing theliquid polypeptide/TBA mixture. In certain embodiments, the polypeptideis an antibody. In some embodiments, the polypeptide is an antibody andthe method comprises preparing a liquid composition comprising theantibody and a sugar at a molar ratio of sugar:antibody of at leastabout 360:1 prior to adding TBA to the liquid composition.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized antibody composition, whereinthe method comprises preparing a liquid composition comprising theantibody, adding tert-butyl alcohol (TBA) to the liquid composition toform a liquid antibody/TBA mixture, freezing the liquid antibody/TBAmixture, lyophilizing the liquid antibody/TBA mixture to form alyophilized antibody composition, and reconstituting the lyophilizedantibody composition with a diluent, wherein the time for reconstitutingthe antibody lyophilized in the presence of TBA is less than the timefor reconstituting the same amount of the same antibody lyophilized inthe absence of TBA. In some embodiments, the amount of TBA added to theliquid polypeptide composition to form a liquid polypeptide/TBA mixtureis between about 0.5% to about 20% by volume. In other embodiments, theamount of TBA added to the liquid polypeptide composition to form theliquid polypeptide/TBA mixture is selected from the group consisting of0.5%, 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, and 20% by volume. Inyet other embodiments, the amount of TBA added to the liquid polypeptidecomposition to form the liquid polypeptide/TBA mixture is between about1% to about 20% by volume, about 1% to about 10% by volume, about 1% toabout 5% by volume, about 5% to about 20% by volume, or about 5% toabout 10% by volume. In some embodiments, the method comprises preparinga liquid composition comprising the antibody and a sugar at a molarratio of sugar:antibody of at least about 360:1 prior to adding TBA tothe liquid composition. In one embodiment, the TBA is added to theliquid composition comprising the polypeptide immediately prior tofreezing the liquid polypeptide/TBA mixture. In one embodiment, theantibody is a monoclonal antibody. In one embodiment, the antibody is ahuman monoclonal antibody. In one embodiment, the antibody is ahumanized monoclonal antibody. In one embodiment, the antibody is achimeric antibody. In one embodiment, the antibody is an antibodyfragment. In one embodiment, the antibody is a bispecific antibody. Inone embodiment, the antibody is an antibody-drug conjugate (e.g., animmunoconjugate).

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized polypeptide composition,wherein the method comprises preparing a liquid composition comprisingthe polypeptide, wherein the concentration of the polypeptide in theliquid composition comprising the polypeptide is between about 10 mg/mlto about 150 mg/ml, adding tert-butyl alcohol (TBA) to the liquidcomposition to form a liquid polypeptide/TBA mixture, freezing theliquid polypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBAmixture to form a lyophilized polypeptide composition, andreconstituting the lyophilized polypeptide composition with a diluent,wherein the time for reconstituting the polypeptide lyophilized in thepresence of TBA is less than the time for reconstituting the same amountof the same polypeptide lyophilized in the absence of TBA. In someembodiments, the concentration of the polypeptide in the liquidcomposition comprising the polypeptide is between about 10 mg/ml toabout 250 mg/ml, between about 25 mg/ml to about 250 mg/ml, betweenabout 50 mg/ml to about 250 mg/ml, between about 100 mg/ml to about 250mg/ml, between about 150 mg/mml to about 250 mg/ml, between about 25mg/ml to about 100 mg/ml, between about 50 mg/ml to about 100 mg/ml,between about 25 mg/ml to about 125 mg/ml, or between about 50 mg/ml toabout 125 mg/ml. In some embodiments, the concentration of thepolypeptide in the liquid composition comprising the polypeptide isabout 25 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70mg/ml, about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 200mg/ml, or about 250 mg/ml. In certain embodiments, the polypeptide is anantibody. In some embodiments, the amount of TBA added to the liquidpolypeptide composition to form a liquid polypeptide/TBA mixture isbetween about 0.5% to about 20% by volume. In some embodiments, thepolypeptide is an antibody and the method comprises preparing a liquidcomposition comprising the antibody and a sugar at a molar ratio ofsugar:antibody of at least about 360:1 prior to adding TBA to the liquidcomposition.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized polypeptide composition,wherein the method comprises preparing a liquid composition comprisingthe polypeptide, wherein the amount of the polypeptide in the liquidcomposition comprising the polypeptide is between about 150 mg to about11 grams, adding tert-butyl alcohol (TBA) to the liquid composition toform a liquid polypeptide/TBA mixture, freezing the liquidpolypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBA mixtureto form a lyophilized polypeptide composition, and reconstituting thelyophilized polypeptide composition with a diluent, wherein the time forreconstituting the polypeptide lyophilized in the presence of TBA isless than the time for reconstituting the same amount of the samepolypeptide lyophilized in the absence of TBA. In some embodiments, theamount of the polypeptide in the liquid composition comprising thepolypeptide is about 150 mg, about 250 mg, about 500 mg, about 750 mg,about 1 gram, about 2 grams, about 3 grams, about 4 grams, about 5grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, about10 grams, or about 11 grams. In some embodiments, the amount of thepolypeptide in the liquid composition comprising the polypeptide isbetween about 150 mg to 11 grams. In some embodiments, the amount of thepolypeptide in the liquid composition comprising the polypeptide isbetween about 150 mg to about 1 gram, between about 500 mg to about 1gram, between about 500 mg to about 5 grams, between about 1 gram toabout 5 grams, between about 1 gram to about 10 grams, or between about5 grams to about 10 grams. In some embodiments, the quantity of thepolypeptide in the liquid composition comprising the polypeptide isgreater than 11 grams. In certain embodiments, the polypeptide is anantibody. In some embodiments, the amount of TBA added to the liquidpolypeptide composition to form a liquid polypeptide/TBA mixture isbetween about 0.5% to about 20% by volume. In some embodiments, thepolypeptide is an antibody and the method comprises preparing a liquidcomposition comprising the antibody and a sugar at a molar ratio ofsugar:antibody of at least about 360:1 prior to adding TBA to the liquidcomposition.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized polypeptide composition,wherein the method comprises preparing a liquid composition comprisingthe polypeptide, adding tert-butyl alcohol (TBA) to the liquidcomposition to form a liquid polypeptide/TBA mixture, freezing theliquid polypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBAmixture to form a lyophilized polypeptide composition, andreconstituting the lyophilized polypeptide composition with a diluent,wherein the time for reconstituting the polypeptide lyophilized in thepresence of TBA is less than the time for reconstituting the same amountof the same polypeptide lyophilized in the absence of TBA, wherein thetime for reconstituting the lyophilized polypeptide composition whichwas lyophilized in the presence of TBA is reduced by about 40% to about95% compared to the time for reconstituting the same amount of the samepolypeptide composition lyophilized in the absence of TBA. In someembodiments, the time for reconstituting the lyophilized polypeptidecomposition which was lyophilized in the presence of TBA is reduced byabout 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, or about 90% to about 95% compared to the time forreconstituting the same amount of the same polypeptide compositionlyophilized in the absence of TBA. In some embodiments, the polypeptideis an antibody and the method comprises preparing a liquid compositioncomprising the antibody and a sugar at a molar ratio of sugar:antibodyof at least about 360:1 prior to adding TBA to the liquid composition.

In some embodiments of the present invention, the liquid polypeptide/TBAmixture is lyophilized in a vial, a syringe (including dual-chambersyringe), or a cartridge. In some embodiments, the liquidpolypeptide/TBA mixture is lyophilized in a glass vial. In someembodiments, the vial is a 6 cc vial, a 10 cc vial, a 15 cc vial, a 20cc vial, a 50 cc vial, or a 100 cc vial. In some embodiments, the glassvial is a 6 cc vial, a 10 cc vial, a 15 cc vial, a 20 cc vial, a 50 ccvial, or a 100 cc vial. In some embodiments, the vials have a capacityor fill volume of 0.1 ml, 0.3 ml, 0.5 ml, 1 ml, 2 ml, 2.5 ml, 5 ml, 10ml, 20 ml, 25 ml, 50 ml, or 100 ml. In some embodiments, the glass vialshave a capacity or fill volume of 0.1 ml, 0.3 ml, 0.5 ml, 1 ml, 2 ml,2.5 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50 ml, or 100 ml.

In some embodiments, reconstituting the lyophilized polypeptidecomposition is performed under conditions in which reconstituting thelyophilized polypeptide composition is performed under a vacuum. In someembodiment, the present invention provides a method for reducing thereconstitution time of a lyophilized polypeptide composition, whereinthe method comprises preparing a liquid composition comprising thepolypeptide, adding tert-butyl alcohol (TBA) to the liquid compositionto form a liquid polypeptide/TBA mixture, freezing the liquidpolypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBA mixtureto form a lyophilized polypeptide composition, and reconstituting thelyophilized polypeptide composition with a diluent, wherein thereconstituting the lyophilized polypeptide composition is performedunder conditions in which the lyophilized polypeptide composition isunder a vacuum, and further wherein the time for reconstituting thepolypeptide lyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same polypeptide lyophilized inthe absence of TBA. In some embodiments, the vacuum is a deep vacuum. Insome embodiments, the vacuum is a partial vacuum. In certainembodiments, the vacuum is less than 100 Torr. In certain embodiments,the vacuum is less than 50 Torr. In certain embodiments, the vacuum isbetween 100 mTorr and 50 Torr. In one embodiment, the vacuum is at 100mTorr. In some embodiments, the polypeptide is an antibody and themethod comprises preparing a liquid composition comprising the antibodyand a sugar at a molar ratio of sugar:antibody of at least about 360:1prior to adding TBA to the liquid composition.

In some embodiments, reconstituting the lyophilized polypeptidecomposition is performed using a shaker, such as a mechanical shaker. Inone embodiment, the present invention provides a method for reducing thereconstitution time of a lyophilized polypeptide composition, whereinthe method comprises preparing a liquid composition comprising thepolypeptide, adding tert-butyl alcohol (TBA) to the liquid compositionto form a liquid polypeptide/TBA mixture, freezing the liquidpolypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBA mixtureto form a lyophilized polypeptide composition, and reconstituting thelyophilized polypeptide composition with a diluent by using a shaker,wherein the time for reconstituting the polypeptide lyophilized in thepresence of TBA is less than the time for reconstituting the same amountof the same polypeptide lyophilized in the absence of TBA. In someembodiments, the vial or container is placed on the shaker. In someembodiments, the polypeptide is an antibody and the method comprisespreparing a liquid composition comprising the antibody and a sugar at amolar ratio of sugar:antibody of at least about 360:1 prior to addingTBA to the liquid composition.

In some embodiments, reconstituting the lyophilized polypeptidecomposition is performed under conditions in which the lyophilizedpolypeptide composition is under a vacuum and using a shaker. In oneembodiment, the present invention provides a method for reducing thereconstitution time of a lyophilized polypeptide composition, whereinthe method comprises preparing a liquid composition comprising thepolypeptide, adding tert-butyl alcohol (TBA) to the liquid compositionto form a liquid polypeptide/TBA mixture, freezing the liquidpolypeptide/TBA mixture, lyophilizing the liquid polypeptide/TBA mixtureto form a lyophilized polypeptide composition, and reconstituting thelyophilized polypeptide composition with a diluent by using a shaker,wherein the reconstituting the lyophilized polypeptide composition isperformed under conditions in which the lyophilized polypeptidecomposition is under a vacuum, and further wherein the time forreconstituting the polypeptide lyophilized in the presence of TBA isless than the time for reconstituting the same amount of the samepolypeptide lyophilized in the absence of TBA. In some embodiments, thevacuum is a deep vacuum. In some embodiments, the vacuum is a partialvacuum. In certain embodiments, the vacuum is less than 100 Torr. Incertain embodiments, the vacuum is less than 50 Torr. In certainembodiments, the vacuum is between 100 mTorr and 50 Torr. In oneembodiment, the vacuum is at 100 mTorr. In some embodiments, thepolypeptide is an antibody and the method comprises preparing a liquidcomposition comprising the antibody and a sugar at a molar ratio ofsugar:antibody of at least about 360:1 prior to adding TBA to the liquidcomposition.

The present invention provides liquid polypeptide formulations suitablefor lyophilization. In some embodiments, the present invention providesliquid polypeptide formulations suitable for lyophilization, wherein theformulation comprises the polypeptide, a diluent, and TBA, wherein thetime for reconstituting the polypeptide lyophilized in the presence ofTBA is less than the time for reconstituting the same amount of the samepolypeptide lyophilized in the absence of TBA. In some embodiments, theamount of TBA in the liquid formulation is between about 0.5-20% byvolume. In some embodiments, the amount of TBA in the liquid formulationis selected from the group consisting of 0.5%, 1%, 2.5%, 5%, 7.5%, 10%,12.5%, 15%, 17.5%, and 20% by volume. In other embodiments, the amountof TBA added to the liquid polypeptide composition to form the liquidpolypeptide/TBA mixture is between about 1% to about 20% by volume,between about 1% to about 10% by volume, between about 1% to about 5% byvolume, between about 5% to about 20% by volume, or between about 5% toabout 10% by volume. In one embodiment, the TBA is added to the liquidcomposition comprising the polypeptide immediately prior to freezing theliquid polypeptide/TBA mixture. In certain embodiments, the polypeptideis an antibody. In some embodiments, the polypeptide is an antibody andthe formulation further comprises a sugar at a molar ratio ofsugar:antibody of at least about 360:1.

The present invention provides a lyophilized polypeptide composition,wherein the lyophilized polypeptide composition is produced by preparinga liquid composition comprising the polypeptide, adding tert-butylalcohol (TBA) to the liquid composition to form a liquid polypeptide/TBAmixture, freezing the liquid polypeptide/TBA mixture, lyophilizing theliquid polypeptide/TBA mixture to form a lyophilized polypeptidecomposition, wherein upon reconstitution of the lyophilized polypeptidecomposition with a diluent, the time for reconstituting the polypeptidelyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same polypeptide lyophilized inthe absence of TBA. In some embodiments, the amount of TBA in the liquidformulation is between about 0.5% to about 20% by volume. In someembodiments, the amount of TBA in the liquid formulation is selectedfrom the group consisting of 0.5%, 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%,17.5%, and 20% by volume. In other embodiments, the amount of TBA addedto the liquid polypeptide composition to form the liquid polypeptide/TBAmixture is between about 1% to about 20% by volume, between about 1% toabout 10% by volume, between about 1% to about 5% by volume, betweenabout 5% to about 20% by volume, or between about 5% to about 10% byvolume. In one embodiment, the TBA is added to the liquid compositioncomprising the polypeptide immediately prior to freezing the liquidpolypeptide/TBA mixture. In certain embodiments, the polypeptide is anantibody. In certain embodiments, the addition of TBA to the polypeptideformulation has no effect on the integrity or stability of thepolypeptide. In some embodiments, the polypeptide is an antibody and thelyophilized polypeptide composition is produced by preparing a liquidcomposition comprising the antibody and a sugar at a molar ratio ofsugar:antibody of at least about 360:1 prior to adding TBA to the liquidcomposition.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized antibody composition, whereinthe method comprises preparing a liquid composition comprising theantibody and a sugar at a molar ratio of sugar:antibody of at leastabout 360:1, adding tert-butyl alcohol (TBA) to the liquid compositionto form a liquid antibody/TBA mixture, wherein the amount of TBA in theliquid antibody/TBA mixture is between about 5%-20% by volume, freezingthe liquid antibody/TBA mixture, lyophilizing the liquid antibody/TBAmixture to form a lyophilized antibody composition, and reconstitutingthe lyophilized antibody composition with a diluent, wherein the timefor reconstituting the antibody lyophilized in the presence of TBA isless than the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA. In some embodiments, theamount of TBA added to the liquid antibody composition to form theliquid antibody/TBA mixture is selected from the group consisting of 5%,7.5%, 10%, 12.5%, 15%, 17.5%, and 20% by volume. In some embodiments,the amount of TBA added to the liquid antibody composition to form theliquid antibody/TBA mixture is about 5%-6% by volume. In yet otherembodiments, the amount of TBA added to the liquid antibody compositionto form the liquid antibody/TBA mixture is between about 5% to about 10%by volume. In one embodiment, the TBA is added to the liquid compositioncomprising the antibody immediately prior to freezing the liquidantibody/TBA mixture. In certain embodiments, the antibody is a fulllength antibody. In one embodiment, the antibody is a monoclonalantibody. In one embodiment, the antibody is a human monoclonalantibody. In one embodiment, the antibody is a humanized monoclonalantibody. In one embodiment, the antibody is a chimeric antibody. In oneembodiment, the antibody is an antibody fragment. In one embodiment, theantibody is an antigen-binding fragment, such as a Fv, Fab, Fab′,Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibodymolecules (e.g. scFv); and multispecific antibodies formed from antibodyfragments. In one embodiment, the antibody is a bispecific antibody. Inone embodiment, the antibody is an antibody-drug conjugate (e.g., animmunoconjugate). In some embodiments, the sugar is sucrose ortrehalose. In certain embodiments, the addition of TBA to the liquidcomposition has no effect on the integrity or stability of the antibody.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized antibody composition, whereinthe method comprises preparing a liquid composition comprising theantibody and a sugar at a molar ratio of sugar:antibody of at leastabout 360:1, wherein the concentration of the antibody in the liquidcomposition comprising the antibody is between about 10 mg/ml to about250 mg/ml, adding tert-butyl alcohol (TBA) to the liquid composition toform a liquid antibody/TBA mixture, wherein the amount of TBA in theliquid antibody/TBA mixture is between about 5%-20% by volume, freezingthe liquid antibody/TBA mixture, lyophilizing the liquid antibody/TBAmixture to form a lyophilized antibody composition, and reconstitutingthe lyophilized antibody composition with a diluent, wherein the timefor reconstituting the antibody lyophilized in the presence of TBA isless than the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA. In some embodiments, theamount of TBA added to the liquid antibody composition to form theliquid antibody/TBA mixture is about 5%-6% by volume. In someembodiments, the concentration of the antibody in the liquid compositioncomprising the antibody is between about 25 mg/ml to about 250 mg/ml,between about 50 mg/ml to about 250 mg/ml, between about 100 mg/ml toabout 250 mg/ml, between about 150 mg/mml to about 250 mg/ml, betweenabout 25 mg/ml to about 100 mg/ml, between about 50 mg/ml to about 100mg/ml, between about 25 mg/ml to about 125 mg/ml, or between about 50mg/ml to about 125 mg/ml. In some embodiments, the concentration of theantibody in the liquid composition comprising the antibody is about 25mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml,about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 200 mg/ml, orabout 250 mg/ml. In some embodiments, the sugar is sucrose or trehalose.In certain embodiments, the addition of TBA to the liquid compositionhas no effect on the integrity or stability of the antibody.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized antibody composition, whereinthe method comprises preparing a liquid composition comprising theantibody and a sugar at a molar ratio of sugar:antibody of at leastabout 360:1, wherein the amount of the antibody in the liquidcomposition comprising the antibody is between about 150 mg to about 11grams, adding tert-butyl alcohol (TBA) to the liquid composition to forma liquid antibody/TBA mixture, wherein the amount of TBA in the liquidantibody/TBA mixture is between about 5%-20% by volume, freezing theliquid antibody/TBA mixture, lyophilizing the liquid antibody/TBAmixture to form a lyophilized antibody composition, and reconstitutingthe lyophilized antibody composition with a diluent, wherein the timefor reconstituting the antibody lyophilized in the presence of TBA isless than the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA. In some embodiments, theamount of TBA added to the liquid antibody composition to form theliquid antibody/TBA mixture is about 5%-6% by volume. In someembodiments, the amount of the antibody in the liquid compositioncomprising the antibody is about 150 mg, about 250 mg, about 500 mg,about 750 mg, about 1 gram, about 2 grams, about 3 grams, about 4 grams,about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9grams, about 10 grams, or about 11 grams. In some embodiments, theamount of the antibody in the liquid composition comprising the antibodyis between about 150 mg to about 1 gram, between about 500 mg to about 1gram, between about 500 mg to about 5 grams, between about 1 gram toabout 5 grams, between about 1 gram to about 10 grams, or between about5 grams to about 10 grams. In some embodiments, the quantity of theantibody in the liquid composition comprising the antibody is greaterthan 11 grams. In some embodiments, the sugar is a sucrose or trehalose.In certain embodiments, the addition of TBA to the liquid compositionhas no effect on the integrity or stability of the antibody.

In one embodiment, the present invention provides a method for reducingthe reconstitution time of a lyophilized antibody composition, whereinthe method comprises preparing a liquid composition comprising theantibody and a sugar at a molar ratio of sugar:antibody of at leastabout 360:1, adding tert-butyl alcohol (TBA) to the liquid compositionto form a liquid antibody/TBA mixture, wherein the amount of TBA in theliquid antibody/TBA mixture is between about 5%-20% by volume, freezingthe liquid antibody/TBA mixture, lyophilizing the liquid antibody/TBAmixture to form a lyophilized antibody composition, and reconstitutingthe lyophilized antibody composition with a diluent, wherein the timefor reconstituting the antibody lyophilized in the presence of TBA isless than the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA, wherein the time forreconstituting the lyophilized antibody composition which waslyophilized in the presence of TBA is reduced by about 40% to about 95%compared to the time for reconstituting the same amount of the sameantibody composition lyophilized in the absence of TBA. In someembodiments, the amount of TBA added to the liquid antibody compositionto form the liquid antibody/TBA mixture is about 5%-6% by volume. Insome embodiments, the time for reconstituting the lyophilized antibodycomposition which was lyophilized in the presence of TBA is reduced byabout 40%, by about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 50% to about 60%, about 60% to about 70%, about 70% toabout 80%, about 80% to about 90%, or about 90% to about 95% compared tothe time for reconstituting the same amount of the same antibodycomposition lyophilized in the absence of TBA. In some embodiments, thesugar is sucrose or trehalose. In certain embodiments, the addition ofTBA to the liquid composition has no effect on the integrity orstability of the antibody.

In some embodiments of the present invention, the liquid antibody/TBAmixture is lyophilized in a vial, a syringe (including dual-chambersyringe), or a cartridge. In some embodiments, the liquid antibody/TBAmixture is lyophilized in a glass vial. In some embodiments, the vial isa 6 cc vial, a 10 cc vial, a 15 cc vial, a 20 cc vial, a 50 cc vial, ora 100 cc vial. In some embodiments, the vials have a capacity or fillvolume of 0.1 ml, 0.3 ml, 0.5 ml, 1 ml, 2 ml, 2.5 ml, 5 ml, 10 ml, 20ml, 25 ml, 50 ml, or 100 ml.

In some embodiments, reconstituting the lyophilized antibody compositionis performed under conditions in which reconstituting the lyophilizedantibody composition is performed under a vacuum. In some embodiment,the present invention provides a method for reducing the reconstitutiontime of a lyophilized antibody composition, wherein the method comprisespreparing a liquid composition comprising the antibody and a sugar at amolar ratio of sugar:antibody of at least about 360:1, adding tert-butylalcohol (TBA) to the liquid composition to form a liquid antibody/TBAmixture, wherein the amount of TBA in the liquid antibody/TBA mixture isbetween about 5%-20% by volume, freezing the liquid antibody/TB Amixture, lyophilizing the liquid antibody/TB A mixture to form alyophilized antibody composition, and reconstituting the lyophilizedantibody composition with a diluent, wherein the reconstituting thelyophilized antibody composition is performed under conditions in whichthe lyophilized antibody composition is under a vacuum, and furtherwherein the time for reconstituting the antibody lyophilized in thepresence of TBA is less than the time for reconstituting the same amountof the same antibody lyophilized in the absence of TBA. In someembodiments, the amount of TBA added to the liquid antibody compositionto form the liquid antibody/TBA mixture is about 5%-6% by volume. Insome embodiments, the vacuum is a deep vacuum. In some embodiments, thevacuum is a partial vacuum. In certain embodiments, the vacuum is lessthan 100 Torr. In certain embodiments, the vacuum is less than 50 Torr.In certain embodiments, the vacuum is between 100 mTorr and 50 Torr. Inone embodiment, the vacuum is at 100 mTorr.

In some embodiments, reconstituting the lyophilized antibody compositionis performed using a shaker, such as a mechanical shaker. In oneembodiment, the present invention provides a method for reducing thereconstitution time of a lyophilized antibody composition, wherein themethod comprises preparing a liquid composition comprising the antibodyand a sugar at a molar ratio of sugar:antibody of at least about 360:1,adding tert-butyl alcohol (TBA) to the liquid composition to form aliquid antibody/TBA mixture, wherein the amount of TBA in the liquidantibody/TBA mixture is between about 5%-20% by volume, freezing theliquid antibody/TBA mixture, lyophilizing the liquid antibody/TBAmixture to form a lyophilized antibody composition, and reconstitutingthe lyophilized antibody composition with a diluent by using a shaker,wherein the time for reconstituting the antibody lyophilized in thepresence of TBA is less than the time for reconstituting the same amountof the same antibody lyophilized in the absence of TBA. In someembodiments, the vial or container is placed on the shaker. In someembodiments, the amount of TBA added to the liquid antibody compositionto form the liquid antibody/TBA mixture is about 5%-6% by volume.

In some embodiments, reconstituting the lyophilized antibody compositionis performed under conditions in which the lyophilized antibodycomposition is under a vacuum and using a shaker. In one embodiment, thepresent invention provides a method for reducing the reconstitution timeof a lyophilized antibody composition, wherein the method comprisespreparing a liquid composition comprising the antibody and a sugar at amolar ratio of sugar:antibody of at least about 360:1, adding tert-butylalcohol (TBA) to the liquid composition to form a liquid antibody/TBAmixture, wherein the amount of TBA in the liquid antibody/TBA mixture isbetween about 5%-20% by volume, freezing the liquid antibody/TB Amixture, lyophilizing the liquid antibody/TB A mixture to form alyophilized antibody composition, and reconstituting the lyophilizedantibody composition with a diluent by using a shaker, wherein thereconstituting the lyophilized antibody composition is performed underconditions in which the lyophilized antibody composition is under avacuum, and further wherein the time for reconstituting the antibodylyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same antibody lyophilized in theabsence of TBA. In some embodiments, the vacuum is a deep vacuum. Insome embodiments, the vacuum is a partial vacuum. In certainembodiments, the vacuum is less than 100 Torr. In certain embodiments,the vacuum is less than 50 Torr. In certain embodiments, the vacuum isbetween 100 mTorr and 50 Torr. In one embodiment, the vacuum is at 100mTorr. In some embodiments, the amount of TBA added to the liquidantibody composition to form the liquid antibody/TBA mixture is about5%-6% by volume.

The steps of the methods of the present disclosure may be performed byone person or entity, or the steps may be performed by multiple peopleor by multiple entities. The steps may be performed by multiple peopleor multiple entities at the direction of one person or one entity. Forexample, one person or entity may prepare the liquid compositioncomprising the polypeptide and a sugar, and a different person or entitymay add TBA to the liquid composition to form a liquid polypeptide/TBAmixture prior to freezing the liquid polypeptide/TBA mixture. In anotherexample, one person or entity may lyophilize the liquid polypeptide/TBAmixture to form a lyophilized polypeptide composition, and a differentperson or entity may reconstitute the lyophilized polypeptidecomposition with a diluent.

Accordingly, in one aspect, the present invention provides a method forreducing the reconstitution time of a lyophilized polypeptide orantibody composition, wherein the method comprises adding tert-butylalcohol (TBA) to a liquid composition comprising the polypeptide orantibody and a sugar at a molar ratio of at least about 360:1 to form aliquid polypeptide or antibody/TBA mixture, wherein the amount of TBA inthe liquid polypeptide or antibody/TBA mixture is about 5%-20% byvolume, freezing the liquid polypeptide or antibody/TBA mixture,lyophilizing the liquid polypeptide or antibody/TBA mixture to form alyophilized polypeptide or antibody composition, and reconstituting thelyophilized polypeptide or antibody composition with a diluent, whereinthe time for reconstituting the polypeptide or antibody lyophilized inthe presence of TBA is less than the time for reconstituting the sameamount of the same polypeptide or antibody lyophilized in the absence ofTBA. In some embodiments, the amount of TBA in the liquid polypeptide orantibody/TBA mixture is about 5%-6% by volume.

In another aspect, the present invention provides a method for reducingthe reconstitution time of a lyophilized polypeptide or antibodycomposition, wherein the method comprises adding tert-butyl alcohol(TBA) to a liquid composition comprising the polypeptide or antibody anda sugar at a molar ratio of at least about 360:1 to form a liquidpolypeptide or antibody/TBA mixture, wherein the amount of TBA in theliquid polypeptide or antibody/TBA mixture is about 5%-20% by volume,freezing the liquid polypeptide or antibody/TB A mixture, andlyophilizing the liquid polypeptide or antibody/TB A mixture to form alyophilized polypeptide or antibody composition, wherein the lyophilizedpolypeptide or antibody composition is subsequently reconstituted with adiluent, wherein the time for reconstituting the polypeptide or antibodylyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same polypeptide or antibodylyophilized in the absence of TBA. In some embodiments, the amount ofTBA in the liquid polypeptide or antibody/TBA mixture is about 5%-6% byvolume.

The present invention provides liquid antibody formulations suitable forlyophilization. In some embodiments, the present invention providesliquid antibody formulations suitable for lyophilization, wherein theformulation comprises the antibody, a sugar, a diluent, and TBA, whereinthe amount of TBA in the liquid formulation is between about 5%-20% byvolume, wherein the amount of sugar in the liquid formulation is at amolar ratio of sugar:antibody of at least about 360:1, and wherein thetime for reconstituting the antibody lyophilized in the presence of TBAis less than the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA. In some embodiments, theamount of TBA in the liquid formulation is selected from the groupconsisting of 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, and 20% by volume. Inother embodiments, the amount of TBA added to the liquid antibodycomposition to form the liquid antibody/TBA mixture is between about 5%to about 10% by volume. In some embodiments, the amount of TBA in theliquid formulation is about 5%-6% by volume. In one embodiment, the TBAis added to the liquid composition comprising the antibody immediatelyprior to freezing the liquid antibody/TBA mixture. In some embodiments,the sugar is sucrose or trehalose. In certain embodiments, the presenceof TBA in the liquid antibody formulation has no effect on the integrityor stability of the antibody.

The present invention provides a lyophilized antibody composition,wherein the lyophilized antibody composition is produced by preparing aliquid composition comprising the antibody and a sugar at a molar ratioof sugar:antibody of at least about 360:1, adding tert-butyl alcohol(TBA) to the liquid composition to form a liquid antibody/TBA mixture,wherein the amount of TBA in the liquid antibody/TBA mixture is betweenabout 5%-20% by volume, freezing the liquid antibody/TBA mixture,lyophilizing the liquid antibody/TBA mixture to form a lyophilizedantibody composition, wherein upon reconstitution of the lyophilizedantibody composition with a diluent, the time for reconstituting theantibody lyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same antibody lyophilized in theabsence of TBA. In some embodiments, the amount of TBA in the liquidformulation is selected from the group consisting of 5%, 7.5%, 10%,12.5%, 15%, 17.5%, and 20% by volume. In other embodiments, the amountof TBA added to the liquid antibody composition to form the liquidantibody/TBA mixture is between about 5% to about 10% by volume. In someembodiments, the amount of TBA added to the liquid antibody compositionto form the liquid antibody/TBA mixture is between about 5% to about 6%by volume. In one embodiment, the TBA is added to the liquid compositioncomprising the antibody immediately prior to freezing the liquidantibody/TBA mixture. In some embodiments, the sugar is sucrose ortrehalose. In certain embodiments, the addition of TBA to the liquidcomposition has no effect on the integrity or stability of the antibodyin the liquid composition or in the lyophilized antibody composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth data showing reconstitution times for a lyophilizedantibody formulation containing 0%, 0.5%, 1%, 5%, and 20% (v/v)tert-butyl alcohol.

FIG. 2 sets forth data showing reconstitution times for a 2.5 mllyophilized antibody formulation containing 0%, 5%, and 10% (v/v)tert-butyl alcohol lyophilized in 6 cc vials.

FIG. 3 sets forth data showing reconstitution times for a 25 mllyophilized antibody formulation containing 0%, 1%, 5%, 10%, and 20%(v/v) tert-butyl alcohol lyophilized in 50 cc vials.

FIG. 4 sets forth data showing the effect of vacuum on reconstitutiontimes for a 60 ml lyophilized antibody formulation containing 0%, 5%,and 10% (v/v) tert-butyl alcohol lyophilized in 100 cc vials.

FIG. 5 sets forth data showing the effect of a shaker on reconstitutiontimes for a 100 ml lyophilized antibody formulation containing 0%, 5%,and 10% (v/v) tert-butyl alcohol lyophilized in 100 cc vials.

FIG. 6 sets forth data showing the effect of vacuum and a shaker onreconstitution times for a 100 ml lyophilized antibody formulationcontaining 0%, 5%, and 10% (v/v) tert-butyl alcohol lyophilized in 100cc vials.

FIG. 7 sets forth data showing the effect of vacuum, shaker, and vacuumand shaker on reconstitution times for a 100 ml lyophilized antibodyformulation containing 0% or 5% (v/v) tert-butyl alcohol in 100 ccvials.

FIGS. 8A and 8B sets forth data showing 7-day stability analysis of anantibody formulation containing 0%, 1%, 5%, 10%, and 20% (v/v)tert-butyl alcohol at either 2-8° C. or 30° C. as measured by sizeexclusion chromatography (SEC).

FIG. 9 sets forth data showing 7-day stability analysis of an antibodyformulation containing 0%, 1%, 5%, 10%, and 20% (v/v) tert-butyl alcoholat either 2-8° C. or 30° C. as measured by IEC.

FIGS. 10A and 10B set forth data showing 4-month stability analysis oflyophilized antibody formulations at 50° C. that were generated frompre-lyophilization liquid antibody mixtures containing 0%, 5%, or 10%(v/v) tert-butyl alcohol. Monomer percentages were measured by SEC. FIG.10A shows results for mAb1, and FIG. 10B shows results for mAb2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, inter alia, polypeptide formulations,lyophilization formulations, methods of making lyophilizationformulations, and methods and formulations useful for reducingreconstitution time of a lyophilized polypeptide.

Definitions

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multi-specific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “antibody-drug conjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, cryo protectant(e.g., sucrose, trehalose), or preservative. A pharmaceuticallyacceptable carrier or diluent includes sterile water for injection.

As used herein, “reconstitution time” and grammatical variations thereofrefer to the amount of time necessary for a lyophilized molecule to bedissolved and/or suspended in a liquid form. For example, reconstitutiontime includes, but is not limited to, the time required for a dried(i.e., lyophilized) pellet or cake of a polypeptide to become suspendedin water or a buffer following lyophilization. A “reduction” or“reducing” or “reduced” reconstitution time and grammatical variationsthereof means less time is required for an amount of a polypeptidelyophilized under a first formulation and/or condition to suspend in aliquid compared with the same amount of the same polypeptide lyophilizedunder a second formulation and/or condition and suspended in the sameliquid.

As used herein, the term “cake” or “lyophilized cake” or “lyo cake”refers to a freeze-dried (i.e., lyophilized) polypeptide composition orformulation.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

Pharmaceutical Formulations

Pharmaceutical formulations of an antibody as described herein areprepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer. In certain embodiments, anantibody formulation of the present invention comprises a liquidantibody composition and TBA, wherein the TBA is added to the liquidantibody composition prior to lyophilization. In some aspects, anantibody formulation of the present invention comprises an amount of TBAin the liquid antibody composition selected from the group consisting of0.5%, 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, and 20% by volume. Inother embodiments, the amount of TBA in the liquid antibody compositionis between about 1% to about 20% by volume, between about 1% to about10% by volume, between about 1% to about 5% by volume, between about 5%to about 20% by volume, or between about 5% to about 10% by volume.

In some embodiments, antibody compositions and formulations of thepresent disclosure comprise the antibody and a sugar at a molar ratio ofsugar:antibody of at least about 360:1. In some embodiments, the molarratio of sugar:antibody is at least about 360:1, at least about 380:1,at least about 400:1, at least about 420:1, at least about 440:1, atleast about 460:1, at least about 480:1, or at least about 500:1,including every value in between these numbers. In some embodiments, theratio is between about 360:1 and about 500:1. The sugar may be sucroseor trehalose or any other sugar known in the art to improve thestability of proteins during lyophilization. As used herein, the weightof trehalose in the formulation for calculating the weight ratio of theantibody to the trehalose is based on the amount of trehalose anhydrous(MW 342.30). If other forms of trehalose (e.g., trehalose dihydrate) areused, the weight of the trehalose in the formulation should be convertedfrom the weight of trehalose dihydrate with the same molarconcentration. When calculating the molar ratio of sugar:antibody, amolecular weight of 150 kDa may be assumed for the antibody.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

The present invention provides, inter alia, polypeptide formulations,lyophilization formulations, and methods for reducing the reconstitutiontime of a lyophilized polypeptide composition. Lyophilizationmethodologies and techniques are well-known in the art. Generally, theprocess of lyophilization is as follows: A dissolved substance (e.g.,biological molecule, polypeptide) is frozen in its final package orstorage container (e.g., vial, syringe, or cartridge) at a lowtemperature (e.g., −60° C.). Lyophilization, or freeze drying, is aprocess in which water is removed from a product after it is frozen andplaced under vacuum, allowing the ice to change directly from solid tovapor without passing through a liquid phase. The process involves threeseparate and interdependent processes: freezing, primary drying(sublimation), and secondary drying (desorption). What results is a drylyophilization cake (i.e., a dry lyo cake), the structure of which,including its density, pore size, total pore volume, and surface areaare controlled by production (e.g., diluent, temperature, vacuumstrength, drying conditions, freezing conditions, etc.). With properstorage, lyophilized biological molecules and polypeptides typicallyremain stable for 2 or 3 years.

Lyophilization containers include, for example, vials, syringes, orcartridges. Numerous lyophilization containers of various sizes arecommercially available, many of which are made of glass. Vial sizes foruse in lyophilization include, but are not limited to, 6 cc vials, 10 ccvials, 15 cc vials, 20 cc vials, 50 cc vials, 100 cc vials, etc.Lyophilization vials of various capacities (or fill volumes) are alsoavailable, including vials having a capacity or fill volume of 0.1 ml,0.3 ml, 0.5 ml, 1 ml, 2 ml, 2.5 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50 ml,100 ml, etc.

The present invention provides a method for reducing reconstitution timeof a lyophilized polypeptide composition, the method comprising: (a)preparing a liquid composition comprising the polypeptide, (b) addingtert-butyl alcohol (TBA) to the liquid composition to form a liquidpolypeptide/TBA mixture, (c) freezing the liquid polypeptide/TBAmixture, (d) lyophilizing the liquid polypeptide/TBA mixture to form alyophilized polypeptide composition (i.e., a lyophilized polypeptidecake), (e) reconstituting the lyophilized polypeptide composition with adiluent, wherein the time for reconstituting the polypeptide lyophilizedin the presence of TBA is less than the time for reconstituting the sameamount of the same polypeptide lyophilized in the absence of TBA.

The present invention provides methods and formulations for reducingreconstitution time of a lyophilized polypeptide composition in whichTBA is added to a liquid composition comprising the polypeptide prior tolyophilization. In certain embodiments, the amount of TBA added to theliquid composition comprising the polypeptide is from about 0.5% byvolume to about 20% by volume, from about 1% by volume to about 20% byvolume, from about 1% by volume to about 10% by volume, from about 1% byvolume to about 5% by volume, from about 5% by volume to about 20% byvolume, from about 5% by volume to about 10% by volume, or from about10% by volume to about 20% by volume.

In other embodiments, the amount of TBA added to the liquid compositioncomprising the polypeptide is about 0.5%, 1%, 2.5%, 5%, 7.5%, 10%,12.5%, 15%, 17.5%, or 20% by volume. In one embodiment, the amount ofTBA added to the liquid composition comprising the polypeptide is about5%-6% by volume. In one embodiment, the amount of TBA added to theliquid composition comprising the polypeptide is about 5% by volume. Inone embodiment, the amount of TBA added to the liquid compositioncomprising the polypeptide is 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%,5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, or 6.2%. Inone embodiment, the amount of TBA added to the liquid compositioncomprising the polypeptide is about 10% by volume. In one embodiment,the amount of TBA added to the liquid composition comprising thepolypeptide is about 20% by volume.

The methods and formulations of the present invention are effective atreducing the reconstitution time of various concentrations of apolypeptide, an in particular, useful and effective at reducing thereconstitution time of high-concentration polypeptide compositions. Forexample, the present invention provides methods and formulationseffective at reducing reconstitution time of a lyophilized polypeptidecomposition by adding TBA to a liquid composition comprising thepolypeptide prior to lyophilization, in which the liquid compositioncomprising the polypeptide has a high polypeptide concentration. Incertain embodiments, the concentration of the polypeptide in the liquidcomposition comprising the polypeptide is between about 10 mg/ml and 250mg/ml. In certain embodiments, the concentration of the polypeptide inthe liquid composition comprising the polypeptide is between about 25mg/ml to about 250 mg/ml, between about 50 mg/ml to about 250 mg/ml,between about 100 mg/ml to about 250 mg/ml, between about 150 mg/mml toabout 250 mg/ml, between about 25 mg/ml to about 100 mg/ml, betweenabout 50 mg/ml to about 100 mg/ml, between about 25 mg/ml to about 125mg/ml, or between about 50 mg/ml to about 125 mg/ml. In someembodiments, the concentration of the polypeptide in the liquidcomposition comprising the polypeptide is about 25 mg/ml, about 40mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 100 mg/ml,about 125 mg/ml, about 150 mg/ml, about 200 mg/ml, or about 250 mg/ml.

The methods and formulations of the present invention are effective atreducing the reconstitution time of various amounts and quantities of apolypeptide. For example, the present invention provides methods andformulations effective at reducing reconstitution time of a lyophilizedpolypeptide composition by adding TBA to a liquid composition comprisingthe polypeptide prior to lyophilization, in which the liquid compositioncomprising the polypeptide has a large quantity or amount of thepolypeptide. In certain embodiments, the amount of the polypeptide inthe liquid composition comprising the polypeptide is between about 150mg to about 11 grams. In certain embodiments, the quantity of thepolypeptide in the liquid composition comprising the polypeptide (andfollowing lyophilization) is about 150 mg, about 250 mg, about 500 mg,about 750 mg, about 1 gram, about 2 grams, about 3 grams, about 4 grams,about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9grams, about 10 grams, or about 11 grams. In some embodiments, thequantity of the polypeptide in the liquid composition comprising thepolypeptide is greater than 11 grams.

Swirling or shaking the lyophilization container can further improve thereconstitution time of a lyophilized polypeptide. In some embodiments ofthe present invention, reconstituting the lyophilized polypeptide isperformed by adding a diluent to the lyophilized polypeptide in thelyophilization container and swirling the container (either periodicallyor continuously) until the lyophilized polypeptide is dissolved orsuspended in the diluent. In other embodiments of the present invention,reconstituting the lyophilized polypeptide is performed by adding adiluent to the lyophilized polypeptide in the lyophilization containerand placing the container on a shaker (e.g., a mechanical shaker) andshaking the lyophilization container until the lyophilized polypeptideis dissolved or suspended in the diluent.

Reconstitution of the polypeptide under vacuum can also improve thereconstitution time of a lyophilized polypeptide. In some embodiments ofthe present invention, the lyophilization container comprising thelyophilized polypeptide is maintained under a vacuum duringreconstitution. In one embodiment, the lyophilization containercomprising the lyophilized polypeptide is under full vacuum duringreconstitution. In certain embodiments, the lyophilization containercomprising the lyophilized polypeptide is at less than 100 Torr duringreconstitution. In certain embodiments, the lyophilization containercomprising the lyophilized polypeptide is at less than 50 Torr duringreconstitution. In certain embodiments, the lyophilization containercomprising the lyophilized polypeptide is between 100 mTorr and 50 Torr.In one embodiment, the lyophilization container comprising thelyophilized polypeptide is at 100 mTorr.

The present invention provides lyophilized polypeptide compositionsproduced by a method of the present invention. In some embodiments, thepresent invention provides a lyophilized polypeptide composition,wherein the lyophilized polypeptide composition is produced by preparinga liquid composition comprising the polypeptide, adding tert-butylalcohol (TBA) to the liquid composition to form a liquid polypeptide/TBAmixture, freezing the liquid polypeptide/TBA mixture, lyophilizing theliquid polypeptide/TBA mixture to form a lyophilized polypeptidecomposition, wherein upon reconstitution of the lyophilized polypeptidecomposition with a diluent, the time for reconstituting the polypeptidelyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same polypeptide lyophilized inthe absence of TBA. In certain embodiments, the lyophilized polypeptidecomposition is a lyophilized cake comprising the polypeptide.

The present invention provides liquid polypeptide compositions producedby a method of the present invention. In some embodiments, the presentinvention provides a liquid polypeptide composition, wherein the liquidpolypeptide composition is produced by preparing a liquid compositioncomprising the polypeptide, and adding tert-butyl alcohol (TBA) to theliquid composition to form a liquid polypeptide/TBA mixture, whereinupon the liquid polypeptide/TBA mixture to form a lyophilizedpolypeptide composition and subsequent reconstitution of the lyophilizedpolypeptide composition with a diluent, the time for reconstituting thepolypeptide lyophilized in the presence of TBA is less than the time forreconstituting the same amount of the same polypeptide lyophilized inthe absence of TBA.

In some embodiments of the present disclosure, the addition of TBA tothe liquid composition comprising an antibody and a sugar at a molarratio of sugar:antibody of at least about 360:1, wherein the amount ofTBA in the liquid antibody/TBA mixture is between about 5%-20% byvolume, has no effect on the integrity or stability of the antibody inthe liquid antibody/TBA mixture, in the lyophilized antibodycomposition, or in the liquid antibody formulation suitable forlyophilization. In such embodiments, the antibody essentially retainsits physical stability and/or chemical stability and/or biologicalactivity upon storage in a liquid composition or in a lyophilizedcomposition. Preferably, the antibody in the compositions andformulations provided herein essentially retains its physical andchemical stability, as well as its biological activity upon storage. Thestorage period is generally selected based on the intended shelf-life ofthe formulation. Various analytical techniques for measuring proteinstability are available in the art and are reviewed in Peptide andProtein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc.,New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993), for example. Stability can be measured at a selectedtemperature for a selected time period.

In certain embodiments, the liquid antibody formulation suitable forlyophilization is stable at about 40° C. for at least about 1, 2, 3, 4,5, 6, 7, 14, 21, 28, or more days. In certain embodiments, the liquidantibody formulation is stable at about 40° C. for at least about 1, 2,3, 4, 5, 6, 7, 8, or more weeks. In certain embodiments, the liquidantibody formulation is stable at about 25° C. for at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or more months. In certain embodiments, the liquid antibodyformulation is stable at about 5° C. for at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ormore months. In certain embodiments, the liquid antibody formulation isstable at about −20° C. or lower for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, or more months. Furthermore, the liquid antibody formulationis preferably stable following freezing (to, e.g., −20° C., −40° C. or−70° C.) and thawing of the liquid antibody formulation, for examplefollowing 1, 2 3, 4, or 5 cycles of freezing and thawing.

In certain embodiments, the lyophilized antibody composition is stableat about 25° C. for at least 1, 2, 3, 4, 5, 6, 7, 8, or more weeks, orfor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or more months. In certain embodiments, thelyophilized antibody composition is stable at about 5° C. for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or more months. In certain embodiments, the lyophilizedantibody composition is stable at about −20° C. or lower for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

Stability can be evaluated qualitatively and/or quantitatively in avariety of different ways, including evaluation of aggregate formation(for example using size exclusion chromatography, by measuringturbidity, and/or by visual inspection); by assessing chargeheterogeneity using cation exchange chromatography, image capillaryisoelectric focusing (icIEF) or capillary zone electrophoresis;amino-terminal or carboxy-terminal sequence analysis; mass spectrometricanalysis; SDS-PAGE analysis to compare reduced and intact antibody;peptide map (for example tryptic or LYS-C) analysis; evaluatingbiological activity or antigen binding function of the antibody; etc.Instability may involve any one or more of: aggregation, deamidation(e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization(e.g. Asp isomeriation), clipping/hydrolysis/fragmentation (e.g. hingeregion fragmentation), succinimide formation, unpaired cysteine(s),N-terminal extension, C-terminal processing, glycosylation differences,etc.

An antibody “retains its physical stability” in a composition orpharmaceutical formulation if it shows no signs or very little ofaggregation, precipitation and/or denaturation upon visual examinationof color and/or clarity, or as measured by methods known in the art,such as, for example, UV light scattering or by size exclusionchromatography.

An antibody “retains its chemical stability” in a composition orpharmaceutical formulation, if the chemical stability at a given time issuch that the antibody is considered to still retain its biologicalactivity as defined below. Chemical stability can be assessed, forexample, by detecting and quantifying chemically altered forms of theantibody. Chemical alteration may involve size modification (e.g.clipping) which can be evaluated using size exclusion chromatography,SDS-PAGE and/or matrix-assisted laser desorptionionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example.Other types of chemical alteration include charge alteration (e.g.occurring as a result of deamidation) which can be evaluated byion-exchange chromatography or icIEF, for example.

An antibody “retains its biological activity” in a composition orpharmaceutical formulation, if the biological activity of the antibodyat a given time is within about 10% (within the errors of the assay) ofthe biological activity exhibited at the time the composition orpharmaceutical formulation was prepared as determined in an antigenbinding assay, for example. Other “biological activity” assays forantibodies are elaborated herein below.

As used herein, “biological activity” of a monoclonal antibody refers tothe ability of the antibody to bind to antigen. It can further includeantibody binding to antigen and resulting in a measurable biologicalresponse which can be measured in vitro or in vivo. Such activity may beantagonistic or agonistic.

Antibodies

Exemplary techniques for producing antibodies which can be formulatedaccording to the present invention are provided herein. In oneembodiment, the antigen to which the antibody binds is a biologicallyimportant protein and administration of the antibody to a mammalsuffering from a disease or disorder can result in a therapeutic benefitin that mammal. However, antibodies directed against non-polypeptideantigens (such as tumor-associated glycolipid antigens; see U.S. Pat.No. 5,091,178) are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g., receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-b; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-b1, TGF-b2, TGF-b3, TGF-b4, orTGF-b5; a tumor necrosis factor (TNF) such as TNF-alpha or TNF-beta;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins; CD proteinssuch as CD3, CD4, CD8, CD19, CD20, CD22 and CD40; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-alpha, -beta, and -gamma; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 andIL-10; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CD1 1a, CD1 1b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such asHER2, HER3 or HER4 receptor; and fragments of any of the above-listedpolypeptides.

Exemplary molecular targets for antibodies encompassed by the presentinvention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22,CD34 and CD40; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; B cell surface antigens, such asCD20 or BR3; a member of the tumor necrosis receptor superfamily,including DRS; prostate stem cell antigen (PSCA); cell adhesionmolecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM,alpha4/beta7 integrin, and alphav/beta3 integrin including either alphaor beta subunits thereof (e.g. anti-CD 1 1a, anti-CD 18 or anti-CD 1 1bantibodies); growth factors such as VEGF as well as receptors therefor;tissue factor (TF); a tumor necrosis factor (TNF) such as TNF-alpha orTNF-beta, alpha interferon (alpha-IFN); an interleukin, such as IL-8;IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor;mp1 receptor; CTLA-4; protein C etc.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

Exemplary antibodies which can be formulated according to the presentinvention include, but are not limited to the following: anti-ErbBantibodies, including anti-HER2 antibodies (e.g. trastuzumab(HERCEPTIN®) or pertuzumab); antibodies that bind to a B-cell surfacemarker, such as CD20 (for example rituximab and humanized2H7/ocrelizumab), CD22, CD40 or BR3; antibodies that bind to IgE,including omalizumab (XOLAIR®) commercially available from Genentech,E26, HAE1, IgE antibody with an amino acid substitution at position 265of an Fc region thereof (US 2004/0191244 A1), Hu-901, an IgE antibody asin WO2004/070011, or antibody that binds the small extracellular segmenton IgE, M1′ (e.g. 47H4v5; see U.S. Pat. No. 8,071,097), see, also,Presta et al, J. Immunol. 151:2623-2632 (1993); InternationalPublication No. WO 95/19181; U.S. Pat. No. 5,714,338, issued Feb. 3,1998; U.S. Pat. No. 5,091,313, issued Feb. 25, 1992; WO 93/04173published Mar. 4, 1993; WO 99/01556 published Jan. 14, 1999; and U.S.Pat. No. 5,714,338; antibodies that bind to vascular endothelial growthfactor (VEGF) (e.g. bevacizumab) or a VEGF receptor; anti-IL-8antibodies (St John et al, Chest, 103:932 (1993), and InternationalPublication No. WO 95/23865); anti-PSCA antibodies (WO01/40309);anti-CD40 antibodies, including S2C6 and humanized variants thereof(WO00/75348); anti-CD1 1a antibodies, including efalizumab (RAPTIVA®)(U.S. Pat. No. 5,622,700, WO 98/23761, Steppe et al, Transplant Intl.4:3-7 (1991), and Hourmant et al, Transplantation 58:377-380 (1994));anti-CD18 antibodies (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, oras in WO 97/26912, published Jul. 31, 1997); anti-Apo-2 receptorantibody (WO 98/51793 published Nov. 19, 1998); anti-TNF-alphaantibodies including cA2 (REMICADE®) and adalimumab (HUMIRA®), CDP571and MAK-195 (Afelimomab) (See, U.S. Pat. No. 5,672,347 issued Sep. 30,1997, Lorenz et al. J. Immunol. 156(4): 1646-1653 (1996), and Dhainautet al. Crit. Care Med. 23(9): 1461-1469 (1995)); anti-Tissue Factor (TF)(European Patent No. 0 420 937 B1 granted Nov. 9, 1994); anti-human α4β7integrin (WO 98/06248 published Feb. 19, 1998); anti-EGFR antibodies,including chimerized or humanized 225 antibody as in WO 96/40210published Dec. 19, 1996; anti-CD3 antibodies, such as OKT3 (U.S. Pat.No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-tac antibodies suchas CHI-621 (SIMULECT®) and (ZENAPAX®) (See U.S. Pat. No. 5,693,762issued Dec. 2, 1997); anti-CD4 antibodies such as the cM-7412 antibody(Choy et al. Arthritis Rheum 39(1):52-56 (1996)); anti-CD52 antibodiessuch as alemtuzumab (CAMPATH-1H®) (Riechmann et al. Nature 332:323-337(1988); anti-Fc receptor antibodies such as the M22 antibody directedagainst FcγRI as in Graziano et al. J. Immunol. 155(10):4996-5002(1995); anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14(Sharkey et al. Cancer Res. 55(23Suppl): 5935s-5945s (1995); antibodiesdirected against breast epithelial cells including huBrE-3, hu-Mc 3 andCHL6 (Ceriani et al. Cancer Res. 55(23): 5852s-5856s (1995); and Richmanet al. Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies thatbind to colon carcinoma cells such as C242 (Litton et al. Eur J.Immunol. 26(1): 1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5 (Elliset al. J. Immunol. 155(2):925-937 (1995)); anti-CD33 antibodies such asHu M195 (Jurcic et al. Cancer Res 55(23 Suppl):5908s-5910s (1995) andCMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide(Juweid et al. Cancer Res 55(23 Suppl):5899s-5907s (1995); anti-EpCAMantibodies such as 17-1 A (PANOREX®); anti-GpIIMIIa antibodies such asabciximab or c7E3 Fab (REOPRO®); anti-RSV antibodies such as MEDI-493(SYNAGIS®); anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodiessuch as PR0542; anti-hepatitis antibodies such as the anti-Hep Bantibody OSTAVIR®; anti-CA 125 antibody OvaRex; anti-idiotypic GD3epitope antibody BEC2; anti-avP3 antibody VITAXIN®; anti-human renalcell carcinoma antibody such as ch-G250; ING-1; anti-human 17-1Aantibody (3622W94); anti-human colorectal tumor antibody (A33);anti-human melanoma antibody R24 directed against GD3 ganglioside;anti-human squamous-cell carcinoma (SF-25); and anti-human leukocyteantigen (HLA) antibodies such as Smart ID 10 and the anti-HLA DRantibody Oncolym (Lym-1); anti-CCRS (PRO 140); ABT-325; ABT-308;ABT-147; anti-beta7 (etrolizumab); anti-HER3/EGFR DAF (DL1 If);anti-interleukin 6 receptor (IL6R) such as tocilizumab (ACTEMRA®);anti-FluA (see WO2014/078268) and anti-Abeta (see WO2003/070760 andWO2008/011348), etc.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Reconstitution of 2.5 ml and 25 ml mAb Cakes with 0-20% TBA

Purified monoclonal antibody (mAb) solutions (mAb1 and mAb2) werediluted to 70 mg/ml in a buffer containing 168 mM sucrose, 20 mMHis-HCl, 0.03% PS20. TBA was warmed above its melting point of 25° C.and then added to the mAb samples as follows. TBA was added directly tothe 70 mg/ml mAb solutions for final TBA concentrations of up to 5%. Forsamples having a final TBA concentration of 10% or 20% (volume/volume),higher initial concentrations of mAb1 (90 mg/mL) and mAb2 (100 mg/mL)were used and diluted to 70 mg/ml protein using 20 mM His buffercontaining 168 mM sucrose and 0.03% PS20 following the addition of theappropriate amount of TBA to obtain 10% TBA or 20% TBA (v/v)concentrations in the final mAb solutions.

The mAb/TBA mixtures were then added to washed and depyrogenated Schotttubing vials: 6 cc vials for 2.5 ml fill; 50 cc vials for 25 ml fill.The vials were stoppered with Daikyo D777-1 20 mm lyo stoppers. Thevials were then loaded into a laboratory freeze-dryer (Lyostar II, FTSSystems, Stone Ridge, N.Y. or Lyostar III, SP Scientific, Stone Ridge,N.Y.).

Lyophilization was performed as follows. For experiments usinguncontrolled nucleation vials were equilibrated at 5° C. for 1 hour. Theshelf temperature was then reduced to −35° C. at a rate of −0.3° C./minand held at this temperature for 6 hours.

For experiments using controlled nucleation, vials were equilibrated at−10° C. for 2 hours. The chamber was pressurized to 28.5 psig withnitrogen for 5 minutes and then depressurized over ˜2 seconds. The shelftemperature was then reduced to −35° C. at a rate of −0.3° C./min andheld at this temperature for 6 hours. All vial samples in a givenexperiment were consolidated in one lyophilizer for drying in order toavoid any possible inter-lyophilizer variability in drying behavior.Primary drying was conducted at a shelf temperature of −10° C. and witha chamber pressure of 100 mTorr until the Pirani gauge converged withthe capacitance manometer. Secondary drying (to decrease the moisture inthe dried cakes to an acceptable level of 0.5-2%) was conducted at ashelf temperature of 20° C. and with a chamber pressure of 100 mTorr for12 hours. Following lyophilization, the vials were stored at 5° C.

Lyophilized monoclonal antibody compositions were reconstituted withsterile water for injection (diluent) in amounts sufficient to producesolutions having mAb protein concentrations identical to the proteinconcentrations prior to lyophilization. 2.2 ml of sterile water forinjection was added to each 6 cc vial which originally contained 2.5 mlfill; 22 ml of sterile water for injection was added to each 50 cc vialwhich originally contained 25 ml fill. Prior to reconstitution, vialsand the sterile water for injection (reconstitution diluent) wereallowed to equilibrate at room temperature.

Reconstitution of mAb1 in 6 cc vials was performed by adding 2.2 mlsterile water for injection and swirled for 5 seconds; after which thevials were placed on an orbital shaker at 60 rpm until the antibodysolution was fully reconstituted (i.e., complete dissolution).Reconstitution of mAb2 in 6 cc vials was performed by adding 2.2 mlsterile water for injection and swirled for 5 seconds; after which thevials were manually swirled for 5 seconds every 10 minutes until theantibody solution was fully reconstituted. Reconstitution of mAb1 in 50cc vials was performed by adding 22 ml sterile water for injection andswirled for 5 seconds; after which the vials were swirled for 5 secondsevery 10 minutes until the antibody solution was fully reconstituted.

Vials containing samples for manual reconstitution were swirled for 5seconds immediately following the addition of diluent and then swirledapproximately every ten minutes until the remaining solid was confirmedto be dissolved by visual inspection. Agitation was stopped occasionallyto visually assess remaining solid.

FIG. 1 shows the effect of different concentrations of TBA onreconstitution time (minutes) of lyophilized mAb1 from the 6 cc vials(2.5 ml fill). As shown in FIG. 1, the addition of 0.5%, 1%, 5%, or 20%TBA to the antibody solution prior to lyophilization resulted in areduction in reconstitution time of mAb1. In particular, the addition of5% TBA reduced reconstitution time of the lyophilized antibody fromgreater than 20 minutes to less than 5 minutes, an approximately 80%reduction in reconstitution time compared to non-TBA treated control.

Table 1 below shows the specific reconstitution times associated withthe above-described experiment for three separate 6 cc vials for eachTBA concentration tested.

TABLE 1 Reconstitution Time (minutes) TBA Standard Amount Vial A Vial BVial C Average Deviation 0% TBA 22.18 20.5 ND 21.34 1.19 0.5% TBA   7.3811.17 13.68 10.74 3.17 1% TBA 6.28 9.4 12.13 9.27 2.93 5% TBA 5.02 4.423.03 4.156 1.02 20% TBA  14.03 13.4 14.03 14.49 1.37

FIG. 2 shows the effect of different concentrations of TBA onreconstitution time (minutes) of lyophilized mAb2 from the 6 cc vials(2.5 ml fill). As shown in FIG. 2, the addition of 5% or 10% TBA to theantibody solution prior to lyophilization resulted in a reduction inreconstitution time of mAb2. In particular, the addition of 5% TBAreduced reconstitution time of the lyophilized antibody from greaterthan 20 minutes to approximately 7.5 minutes.

FIG. 3 shows the effect of different concentrations of TBA onreconstitution time (minutes) of lyophilized mAb1 from the 50 cc vials(25 ml fill). As shown in FIG. 3, the addition of 1%, 5%, 10%, or 20%TBA to the antibody solution prior to lyophilization resulted in areduction in reconstitution time of mAb1. In particular, the addition of5% TBA reduced reconstitution time of the lyophilized antibody fromapproximately 70 minutes to approximately 27 minutes.

Taken together, these results showed that addition of TBA to an antibodysolution to a prior to lyophilization results in a reduction inreconstitution time. Additionally, these results showed that 5% TBAprovides the most reduction in reconstitution time compared to thatobtained with 1% TBA, 10% TBA, or 20% TBA.

Example 2. Reconstitution with TBA and Vacuum

The effect on reconstitution time of lyophilized mAb1 under partialvacuum vs full vacuum in combination with TBA was examined as follows.For this study, 100 cc vials were used, containing 60 ml fill volume ofmAb1 at 70 mg/ml. TBA was added to the mAb1 solutions to a finalconcentration of either 5% or 10% TBA. Following lyophilization, vialswere backfilled to either 100 mTorr (vacuum) or 200 Torr (partial vacuumcontrol).

Lyophilized mAb1 in the 100 cc vials was reconstituted to the original60 ml fill volume by addition of sterile water for injection, swirledfor 5 seconds, after which the vials were swirled for 5 seconds every 10minutes until the antibody solution was fully reconstituted (i.e.,complete dissolution).

FIG. 4 shows the effect of different concentrations of TBA (5% and 10%TBA) on reconstitution time of lyophilized mAb1 in 100 cc vials undertwo different vacuum conditions (either 100 mTorr or 200 Torr). As shownin FIG. 4, the addition of either 5% TBA or 10% TBA to the antibodysolution prior to lyophilization resulted in a reduction inreconstitution time of mAb1 in the 100 cc vials with 60 ml fill volume,consistent with the results obtained with either 6 cc or 50 cc vialscontaining either 2.5 ml fill volume or 25 ml fill volume, respectively.In particular, under 200 Torr conditions, the addition of 5% TBA or 10%TBA reduced reconstitution time from greater than 110 minutes with noTBA to approximately 60 minutes with 5% TBA and to approximately 80minutes with 10% TBA.

When reconstitutions were performed under 100 mTorr conditions,reconstitution times were greatly reduced. In particular, under 100mTorr conditions, the addition of 5% TBA or 10% TBA reducedreconstitution times from approximately 40 minutes with no TBA toapproximately 20 minutes with 5% TBA and approximately 40 minutes with10% TBA.

Taken together, these results showed that combining TBA with vacuumshowed a greater reduction in reconstitution time compared to using TBAor vacuum alone.

Example 3. Reconstitution with TBA Under Various Shaker Conditions

The effect on reconstitution time of lyophilized mAb1 whenreconstitution was performed with or without the aid of a shaker incombination with TBA was examined as follows. For this study, 100 ccvials were used, containing 60 ml fill volume of mAb1 at 70 mg/ml. TBAwas added to the mAb1 solutions to a final concentration of either 5% or10% TBA.

Lyophilized mAb1 in the 100 cc vials was reconstituted to the original60 ml fill volume by addition of sterile water for injection. Vialscontaining samples for manual reconstitution were swirled for 5 secondsimmediately following the addition of diluent and then swirledapproximately every ten minutes until the remaining solid was confirmedto be dissolved by visual inspection. Vials containing samples forreconstitution by shaker were agitated with a Novartis/Genentech 500 rpmXolair Shaker (South San Francisco, Calif.) following the addition ofdiluent. Agitation was stopped occasionally to visually assess remainingsolid.

FIG. 5 shows the effect of different concentrations of TBA (5% and 10%TBA) on reconstitution time of lyophilized mAb1 in 100 cc vials whenreconstituted with or without the use of a shaker. As shown in FIG. 5,the addition of either 5% TBA or 10% TBA to the antibody solution priorto lyophilization resulted in a reduction in reconstitution time of mAb1in the 100 cc vials with 60 ml fill volume. In particular, under control(no shaker) conditions, the addition of TBA prior to lyophilizationreduced reconstitution time from greater than 110 minutes with no TBA toapproximately 60 minutes with 5% TBA and to approximately 80 minuteswith 10% TBA.

When reconstitution was performed using a shaker at 500 rpm,reconstitution times were greatly reduced. In particular, whenreconstitution was performed with a shaker, the addition of TBA reducedreconstitution times from approximately 18 minutes with no TBA toapproximately 10 minutes with either 5% TBA or 10% TBA.

Taken together, these results showed that combining TBA with shakershowed a greater reduction in reconstitution time compared to using TBAalone.

Example 4. Reconstitution with TBA, Vacuum, and Shaker

The effect on reconstitution time of lyophilized mAb1 whenreconstitution was performed under vacuum (100 mTorr) with the aid of ashaker in combination with TBA was examined as follows. For this study,100 cc vials were used, containing 60 ml fill volume of mAb1 at 70mg/ml. TBA was added to the mAb1 solutions to a final concentration ofeither 5% or 10% TBA. Vacuum and shaker conditions were as describedabove in Example 3.

FIG. 6 shows the effect of different concentrations of TBA (5% and 10%TBA) on reconstitution time of lyophilized mAb1 in 100 cc vials whenreconstituted under 100 mTorr with the use of a shaker. As shown in FIG.6, reconstitution took approximately 110 minutes for 100 cc vialscontaining 60 ml fill with no TBA, no vacuum, and no shaker (swirlingfor 5 seconds every 10 minutes). However, the addition of vacuum, TBA,and shaker dramatically reduced reconstitution times, to less than 10minutes; the combination of these three parameters reducedreconstitution time by over 95%.

Using a combination of 5% TBA, vacuum, and shaker, the reconstitutiontime for 4.2 gm of protein was reduced from an average of 115 minutes to5 minutes, allowing for the preparation of high-dose biomoleculeformulations having rapid reconstitution times.

FIG. 7 shows the effect of vacuum, shaker, and vacuum and shaker onreconstitution times for a 100 ml lyophilized antibody formulationcontaining 0% or 5% (v/v) tert-butyl alcohol in 100 cc vials.

Example 5. Liquid Stability Studies

Stability studies to examine the effects of TBA on mAb1 stability inliquid were performed. mAb1 stability was measured by size exclusionchromatography (SEC) and by ion exchange chromatography (IEC). mAb1 (70mg/ml) was added to 50 cc vials (25 ml fill) containing 0% TBA, 1% TBA,5% TBA, 10% TBA, or 20% TBA. Vials were then maintained at 2-8° C. or30° C. for 7 days, during which the stability of the antibody wasdetermined at day 0, day 3, and day 7 for each condition by SEC and IECanalysis.

SEC analysis was performed using an Agilent 1100 or 1200 HPLC (SantaClara, Calif.) with a Tosoh Biosciences TSK gel 7.8 mm ID×30 cm L column(South San Francisco, Calif.). IEC analysis was performed using anAgilent 1200 HPLC with a Dionex ProPac WCX-10 BioLC™ 4×250 mm Analyticalcolumn (Sunnyvale, Calif.).

As shown in FIGS. 8A and 8B, mAb1 maintained good stability uponaddition of various concentrations of TBA over the course of 7 days ateither 2-8° C. or 30° C., as SEC analysis showed maintenance of highpercentage of the monomer peak over the course of 7 days. FIG. 8B showsan expanded version of the graph shown in FIG. 8A (that is, FIG. 8Bshows the data of FIG. 8A without including the data associated with 20%TBA at 30° C.).

As shown in FIG. 9, mAb1 maintained good stability upon addition ofvarious concentrations of TBA over the course of 7 days at either 2-8°C. or 30° C., as IEC analysis showed maintenance of main peak comparableto controls in all cases over the course of 7 days.

Taken together, these results showed that at TBA concentrations below20% (e.g., 1%, 5%, 10% TBA), stability of the antibody is maintained forat least 7 days; some loss of stability, however, was observed at 20%TBA conditions.

Example 6. Moisture Analysis

The effect of TBA addition to mAb1 or mAb2 prior to lyophilization onthe moisture levels of the dried mAbs following lyophilization wasexamined mAb1 and mAb2 (both 70 mg/ml) were added to 6 cc vials asdescribed above. TBA was added to the mAb solutions to a finalconcentration of 0%, 5%, or 10% TBA. The samples were then lyophilizedas described above. Following lyophilization, the moisture content ofthe lyophilized samples (i.e., solid-state samples) was determined usinga Mettler Toledo DL31 Volumetric Karl Fischer Titrator (Columbus, Ohio).

As shown in Table 2 below, the addition of TBA does not affect themoisture levels of the lyophilized antibody preparations.

TABLE 2 Antibody TBA amount Moisture (percent) mAb1 0% TBA 0.80% 5% TBA0.81% 10% TBA  0.77% mAb2 0% TBA 0.78% 5% TBA 0.71% 10% TBA  0.78%

Moisture content was further examined for mAb1 (40 mg/ml in 50 cc vials)with various concentrations of TBA (0%, 5%, 10%) under uncontrollednucleation (UCN) and controlled nucleation (CN). As shown in Table 3below, the addition of TBA does not affect the moisture levels ofantibody preparations lyophilized using standard freeze technique(uncontrolled nucleation). The addition of TBA decreases the moisturecontent of antibody preparations lyophilized using controlled nucleation

TABLE 3 Nucleation condition TBA amount Moisture (percent) UCN 0% TBA0.67% 5% TBA 0.65% 10% TBA  0.62% CN 0% TBA 0.97% 5% TBA 0.44% 10% TBA 0.46%

The addition of TBA to the monoclonal antibody preparation as shown inboth Table 1 and Table 2 show that the addition of TBA does not havemuch effect on the moisture levels in UCN cakes and decreases moisturelevels in CN cakes.

Example 7. BET Analysis to Determine Specific Surface Area

The effect of various concentrations of TBA on specific surface area ofthe lyophilized antibody cake structure was examined mAb1 and mAb2 wereadded to 6 cc vials at 70 mg/ml with 0%, 5%, or 10% TBA and lyophilizedas described above. Specific surface area measurement was performed witha Quantachrome Quadrasorb Evo Automated Surface Area and Pore SizeAnalyzer (Boynton Beach, Fla.). Samples were crushed in a glove box withrelative humidity <3% and loaded into tared bulbs. A sample size ofapproximately 100 mg was used. Desorption of residual water wasperformed at 40° C. with a strong vacuum for at least 3 hours.Multipoint Brunauer-Emmett-Teller (BET) analysis was then performed withrelative pressures P/Po of 0.05 to 0.24 using Krypton as the adsorbate.

Table 4 below shows the results of the specific surface area (SSA)determinations, presented as m²/gram. As shown in Table 4 below, theaddition of TBA to the antibody preparations prior to lyophilizationresulted in an increase in surface area of the lyophilized cakes.

TABLE 4 Antibody TBA amount SA (m²/g) mAb1 0% TBA 0.330 5% TBA 2.262 10%TBA  6.124 mAb2 0% TBA 0.525 5% TBA 1.369 10% TBA  2.835

Surface area was further examined using BET analysis for mAb1 (40 mg/mlin 50 cc vials) with various concentrations of TBA (0%, 5%, 10%) withuncontrolled nucleation (UCN) and controlled nucleation (CN). The poresize for the mAb1 cakes with uncontrolled nucleation (UCN) andcontrolled nucleation (CN) is shown below in Table 5.

TABLE 5 Nucleation condition TBA amount SA (m²/g) UCN 0% TBA 0.696 5%TBA 3.668 10% TBA  2.906 CN 0% TBA 0.488 5% TBA 3.281 10% TBA  7.794

BET analysis suggested that the addition of TBA resulted in differencesin pore size and the lyophilized cake structure, possibly due to TBAforming vertical pores as it sublimates during lyophilization. (SeeNail, S L et al. Freeze-Drying of tert-Butanol/Water Cosolvent Systems:A Case Report on Formation of a Friable Freeze-Dried Powder ofTobramycin Sulfate. Journal of Pharmaceutical Sciences. Vol. 91, No. 4.April 2002. American Pharmacists Association.)

Example 8. Lyophilized Antibody Stability Studies

Stability studies to examine the effects of TBA in thepre-lyophilization liquid antibody/TBA mixture on the stability of theantibody in the subsequently lyophilized antibody composition wereperformed. Stability was measured by size exclusion chromatography(SEC).

6 cc vials were filled with 2.5 mL of formulations containing 70 mg/mLantibody (mAb1 or mAb2), 168 mM sucrose, 20 mM Histidine HCl at pH 5.8,0.028% polysorbate 20, and 0, 5, or 10% (v/v) TBA. The vials werelyophilized with a conservative lyophilization cycle and incubated at50° C. for over four months. Samples were taken monthly and put at −20°C. until batch analysis by size exclusion chromatography. FIG. 10A showsthe percentage monomer maintained in the lyophilized mAb1 compositionsat each monthly timepoint. FIG. 10B shows the results for thelyophilized mAb2 compositions.

The results show that the accelerated degradation of the lyophilized mAbat 50° C. is still within the typical and acceptable range forlyophilized mAbs when 5% TBA is present in the pre-lyophilization liquidantibody/TBA mixture. As described above, this level of TBA was alsoshown to minimize the reconstitution time of the lyophilized mAb. Thus,adding 5% TBA to the pre-lyophilization liquid antibody formulation canprovide an advantage for high dose lyophilized mAb products that requirelarge (e.g. 50 or 100 cc) vials, and where reconstitution times can besignificantly longer compared to smaller volume vials.

What is claimed is:
 1. A liquid antibody formulation suitable forlyophilization, wherein the formulation comprises the antibody, a sugar,a diluent, and TBA, wherein the amount of TBA in the liquid formulationis about 5%-6% by volume, wherein the amount of sugar in the liquidformulation is at a molar ratio of sugar:antibody of at least about360:1, and wherein the time for reconstituting the antibody lyophilizedin the presence of TBA is less than the time for reconstituting the sameamount of the same antibody lyophilized in the absence of TBA.
 2. Theliquid antibody formulation of claim 1, wherein the amount of TBA in theliquid formulation is about 5% by volume.
 3. The liquid antibodyformulation of claim 1, wherein the molar ratio of sugar:antibody isbetween about 360:1 to about 500:1.
 4. The liquid antibody formulationof claim 1, wherein the antibody is a full length antibody.
 5. Theliquid antibody formulation of claim 1, wherein the concentration of theantibody in the liquid formulation is between about 10 mg/ml to about250 mg/ml.
 6. The liquid antibody formulation of claim 1, wherein theamount of the antibody in the liquid formulation is between about 150mg-11 grams.
 7. The liquid antibody formulation of claim 1, wherein thesugar is sucrose or trehalose.
 8. The liquid antibody formulation ofclaim 1, wherein the time for reconstituting the antibody lyophilized inthe presence of TBA is reduced by about 40-95% compared to the time forreconstituting the same amount of the same antibody lyophilized in theabsence of TBA.
 9. The liquid antibody formulation of claim 1, whereinthe time for reconstituting the antibody lyophilized in the presence ofTBA is reduced by at least 50% compared to the time for reconstitutingthe same amount of the same antibody lyophilized in the absence of TBA.10. A lyophilized antibody composition, wherein the lyophilized antibodycomposition is produced by preparing a liquid composition comprising theantibody and a sugar at a molar ratio of sugar:antibody of at leastabout 360:1, adding tert-butyl alcohol (TBA) to the liquid compositionto form a liquid antibody/TBA mixture, wherein the amount of TBA in theliquid composition is about 5%-6% by volume, freezing the liquidantibody/TBA mixture, and lyophilizing the liquid antibody/TBA mixtureto form a lyophilized antibody composition, wherein upon reconstitutionof the lyophilized antibody composition with a diluent, the time forreconstituting the antibody lyophilized in the presence of TBA is lessthan the time for reconstituting the same amount of the same antibodylyophilized in the absence of TBA.
 11. The lyophilized antibodycomposition of claim 10, wherein the amount of TBA in the liquidcomposition is about 5% by volume.
 12. The lyophilized antibodycomposition of claim 10, wherein the molar ratio of sugar:antibody inthe liquid composition is between about 360:1 to about 500:1.
 13. Thelyophilized antibody composition of claim 10, wherein the amount of TBAin the liquid composition is about 5% by volume.
 14. The lyophilizedantibody composition of claim 10, wherein the molar ratio ofsugar:antibody in the liquid composition is between about 360:1 to about500:1.
 15. The lyophilized antibody composition of claim 10, wherein TBAis added to the liquid composition comprising the antibody immediatelyprior to freezing the liquid antibody/TBA mixture.
 16. The lyophilizedantibody composition of claim 10, wherein the antibody is a full lengthantibody.
 17. The lyophilized antibody composition of claim 10, whereinthe concentration of the antibody in the liquid composition is betweenabout 10 mg/ml to about 250 mg/ml.
 18. The lyophilized antibodycomposition of claim 10, wherein the amount of the antibody in theliquid composition is between about 150 mg-11 grams.
 19. The lyophilizedantibody composition of claim 10, wherein the sugar in the liquidcomposition is sucrose or trehalose.
 20. The lyophilized antibodycomposition of claim 10, wherein upon reconstitution of the lyophilizedantibody composition with a diluent, the time for reconstituting theantibody lyophilized in the presence of TBA is reduced by about 40-95%compared to the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA.
 21. The lyophilized antibodycomposition of claim 10, wherein upon reconstitution of the lyophilizedantibody composition with a diluent, the time for reconstituting theantibody lyophilized in the presence of TBA is reduced by at least 50%compared to the time for reconstituting the same amount of the sameantibody lyophilized in the absence of TBA.